The quantitation of analytes in sample body fluids by enzymatic methods is a fairly recent phenomenon. The basic procedure involves determining the sample "blank" by mixing a sample containing the analyte with the enzyme substrates known to be used for the quantitation of that particular analyte. Substrate specific enzymes are then added to the reaction mixture. The enzymatic conversion of the substrates and analyte results in a change in the reaction composition which can be quantitated by various methods which measure the change in absorbance due to the action of substrate specific enzymes on the substrates. This change in absorbance is then correlated with the concentration of analyte in the sample.
For example, the enzymatic quantitation of total CO.sub.2 in serum or plasma involves mixing a sample containing CO.sub.2 with the substrate phosphoenolpyruvate (PEP). After a blank reading is taken, the substrate specific enzyme phosphoenolpyruvate carboxylase (PEPC) is added causing the conversion of PEP to oxaloacetate (OAA) and phosphate according to the following reaction: ##STR1##
The OAA produced is then correlated with the concentration of CO.sub.2 in the sample by various methods. For example, in Wilson et al., Clinical Chemistry, 19:640 (1973) and Munson et al., Clinical Chemistry, 20:872 (1974), both of which are hereby incorporated by reference, the OAA is simultaneously coupled with reduced nicotinamide adenine dinucleotide (NADH) and malate dehydrogenase (MDH) such that the amount of NADH oxidized is directly proportional to the CO.sub.2 in sample. In Norris, et al., Clinical Chemistry, 21:8, 1093 (1975), also incorporated by reference, the OAA is quantitated by reaction with the diazonium salt of Fast Violet B. One principle disadvantage of enzymatic methods for CO.sub.2 is that they are very sensitive to interference from atmospheric CO.sub.2, especially at the alkaline pH required for optimum reaction and stability of NADH.
Plasma ammonia may also be determined enzymatically according to the method of Van Anken et al., Clinical Chemica Acta, 56:151 (1974) which is also incorporated by reference. This method is based upon the following reaction: ##STR2##
The NADP produced is proportional to the concentration of ammonia in the sample and may be quantitated by measuring the change in absorbance due to the oxidation of the coenzyme NADPH to NADP.sup.+.
The enzymatic reagents in this method are also very unstable, particularly the coenzyme NADPH.